Expression of Multicolor Fluorescent Fusion Proteins in Zebrafish Cell Cultures: A Versatile Tool in Cell Biology

2002 ◽  
pp. 373-380 ◽  
Author(s):  
C. K. D. Breek ◽  
F. Van Iren ◽  
S. E. Wijting ◽  
N. Stuurman ◽  
H. P. Spaink
Keyword(s):  
Cell Cultures ◽  
Fusion Proteins ◽  
Cell Biology ◽  
Versatile Tool

scholarly journals Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus

2021 ◽  
Author(s):  
Vishal Shinde ◽  
Nara Sobreira ◽  
Elizabeth S Wohler ◽  
George Maiti ◽  
Nan Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Cultures ◽  
Cell Biology ◽  
Stromal Cell ◽  
Primary Cilia ◽  
Single Amino Acid ◽  
European Ancestry ◽  
Base Substitution ◽  
Amino Acid Substitutions ◽  
Functional Link

Abstract Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

scholarly journals Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

2005 ◽  
Vol 73 (6) ◽  
pp. 3627-3635 ◽  
Author(s):  
Juliette K. Tinker ◽  
Jarrod L. Erbe ◽  
Randall K. Holmes
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Fusion Proteins ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Secretory Diarrhea ◽  
E Coli ◽  
Twin Arginine Translocation ◽  
Heat Labile

ABSTRACT Cholera toxin (CT) is an AB5 toxin responsible for the profuse secretory diarrhea resulting from Vibrio cholerae infection. CT consists of a pentameric, receptor-binding B subunit (CTB) and a monomeric A subunit (CTA) that has latent enzymatic activity. In addition to its enterotoxicity, CT has potent mucosal adjuvant activity and can also function as a carrier molecule with many potential applications in cell biology. In earlier studies, the toxic CTA1 domain was replaced by several other antigenic protein domains to produce holotoxin-like chimeras for use as potential mucosal vaccines. In the present study we utilized the twin arginine translocation (tat) system to produce fluorescent CT chimeras, as well as fluorescent chimeras of Escherichia coli heat-labile toxins LTI and LTIIb. Fusion proteins containing either green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP) and the A2 domain of CT, LTI, or LTIIb were transported to the periplasm of E. coli by the tat system, and the corresponding B polypeptides of CT, LTI, and LTIIb were transported to the periplasm by the sec system. The fluorescent fusion proteins were shown to assemble spontaneously and efficiently with the corresponding B polypeptides in the periplasm to form chimeric holotoxin-like molecules, and these chimeras bound to and entered cultured cells in a manner similar to native CT, LTI, or LTIIb. The GFP and mRFP derivatives of CT, LT, and LTIIb developed here are useful tools for studies on the cell biology of trafficking of the CT/LT family of bacterial enterotoxins. In addition, these constructs provide proof in principle for the development of novel chimeric CT-like or LT-like vaccine candidates containing CTA2 fusion proteins that cannot be delivered to the periplasm of E. coli by use of the sec secretion pathway.

scholarly journals Spheroids as a Type of Three-Dimensional Cell Cultures—Examples of Methods of Preparation and the Most Important Application

2020 ◽  
Vol 21 (17) ◽  
pp. 6225 ◽  
Author(s):  
Kamila Białkowska ◽  
Piotr Komorowski ◽  
Maria Bryszewska ◽  
Katarzyna Miłowska
Keyword(s):  
Cell Cultures ◽  
Cell Biology ◽  
Disease Modeling ◽  
Three Dimensional ◽  
3D Culture ◽  
Matrix Interactions ◽  
Vitro Model ◽  
Extracellular Matrix Interactions ◽  
Natural Cell

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell–cell and cell–extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications.

scholarly journals Ubiquitin fusion proteins in algae: implications for cell biology and the spread of photosynthesis

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Shannon J. Sibbald ◽  
Julia F. Hopkins ◽  
Gina V. Filloramo ◽  
John M. Archibald
Keyword(s):  
Fusion Proteins ◽  
Cell Biology

Ratiometric Nanoviscometers: Applications for Measuring Cellular Physical Properties in 3D Cultures

2020 ◽  
Vol 25 (3) ◽  
pp. 234-246
Author(s):  
Charles McRae White ◽  
Mark A. Haidekker ◽  
William S. Kisaalita
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Cell Biology ◽  
Low Cost ◽  
3D Cell Culture ◽  
Biomechanical Properties ◽  
Practical Applications ◽  
3D Cell Cultures

New insights into the biomechanical properties of cells are revealing the importance of these properties and how they relate to underlying molecular, architectural, and behavioral changes associated with cell state and disease processes. However, the current understanding of how these in vitro biomechanical properties are associated with in vivo processes has been developed based on the traditional monolayer (two-dimensional [2D]) cell culture, which traditionally has not translated well to the three-dimensional (3D) cell culture and in vivo function. Many gold standard methods and tools used to observe the biomechanical properties of 2D cell cultures cannot be used with 3D cell cultures. Fluorescent molecules can respond to external factors almost instantaneously and require relatively low-cost instrumentation. In this review, we provide the background on fluorescent molecular rotors, which are attractive tools due to the relationship of their emission quantum yield with environmental microviscosity. We make the case for their use in both 2D and 3D cell cultures and speculate on their fundamental and practical applications in cell biology.

Cell lines and primary cell cultures in the study of bone cell biology

2004 ◽  
Vol 228 (1-2) ◽  
pp. 79-102 ◽  
Author(s):  
Vicky Kartsogiannis ◽  
Kong Wah Ng
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Cell Biology ◽  
Bone Cell ◽  
Primary Cell ◽  
Primary Cell Cultures

Purification of GFP fusion proteins from transgenic plant cell cultures

2006 ◽  
Vol 49 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Gabriel D. Peckham ◽  
Robert C. Bugos ◽  
Wei Wen Su
Keyword(s):  
Transgenic Plant ◽  
Plant Cell ◽  
Cell Cultures ◽  
Fusion Proteins ◽  
Plant Cell Cultures ◽  
Transgenic Plant Cell Cultures

scholarly journals LIPID COMPOSITION OF CALLUSES CELL NUCLEI OF PLANTS OF DIFFERENT SYSTEMATIC GROUPS

2018 ◽  
Vol 63 (3) ◽  
pp. 335-338
Author(s):  
V. N. Reshetnikov ◽  
O. V. Chizhik
Keyword(s):  
Comparative Analysis ◽  
Cell Cultures ◽  
Metabolic Activity ◽  
Lipid Composition ◽  
Cell Biology ◽  
Neutral Lipids ◽  
Phospholipid Content ◽  
Winter Rye ◽  
Cell Nuclei ◽  
Active Proliferation

The study revealed the results of the comparative analysis of the deoxyribonucleoproteid complex (chromatin) lipid composition in the callus and explant nuclei of plants, belonging to different systematic groups (winter rye, potatoes, peas). It provided the data on the phospho- and neutral lipid composition of the investigated plants and showed that the phospholipid content in the explants from dormant winter rye seed embryos and potato stems, having low metabolic activity, is significantly higher than in the calluses in the stage of active proliferation. Both winter rye and potato calluses tend to have a lower share of neutral lipids, including sterols, in their composition. The revealed factors can be used as markers of metabolic activity of the plant cell biology objects (callus and cell cultures). 

scholarly journals zGrad is a nanobody-based degron system that inactivates proteins in zebrafish

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Naoya Yamaguchi ◽  
Tugba Colak-Champollion ◽  
Holger Knaut
Keyword(s):  
Rna Interference ◽  
Protein Function ◽  
Fusion Proteins ◽  
Gene Activity ◽  
Modern Biology ◽  
Tissue Specific ◽  
Inducible Promoters ◽  
Protein Depletion ◽  
Protein Inactivation ◽  
Versatile Tool

The analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane and cytosolic proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity, while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.

scholarly journals Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures

2020 ◽  
Author(s):  
Michael Laue ◽  
Anne Kauter ◽  
Tobias Hoffmann ◽  
Lars Möller ◽  
Janine Michel ◽  
...  
Keyword(s):  
Vero Cell ◽  
Cell Cultures ◽  
Cell Biology ◽  
Electron Tomography ◽  
Ultrastructural Level ◽  
Lower Productivity ◽  
Fundamental Information ◽  
Qualitative Finding ◽  
Section Electron ◽  
Morphology And Morphometry

AbstractSARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.

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